Graduate Group in Biochemistry and Molecular Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania.Department of Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.

Department of Physiology, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.Yale Cancer Biology Institute, West Haven, CT, and Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut.

Article Figures & Data

Figures

Necitumumab binds to and inhibits EGFR harboring the S468R cetuximab-resistant mutation. A, Confocal imaging of HeLa cells expressing EGFR-eGFP and EGFR-S468R-eGFP stained with Alexa647-labeled antibodies (left, gray scale). Right panels show an overlay of the Alexa647 and GFP fluorescence. B, Binding of the same antibodies to EGFR and EGFR-S468R–expressing HeLa cells analyzed by flow cytometry. The MFI values are plotted over a concentration range from 0.01 to 67 nmol/L of cetuximab (red), necitumumab (blue), or control IgG (black). There is negligible binding of cetuximab or necitumumab to parental HeLa cells over this concentration range (Supplementary Fig. S1C). Plots are derived from three technical replicates, and are representative of data from three independent experiments. C, Normalized GFP MFI signal for HeLa cells expressing EGFR-eGFP or EGFR-S469R-eGFP following 48-hour preincubation with the indicated concentrations of cetuximab (red), necitumumab (blue), or IgG (black). MFI values are normalized to the EGFR-GFP signal with no antibody treatment. Plots are from two technical replicates and representative of data from two independent experiments. D, Effect of antibody pretreatment (1,000 nmol/L) on EGFR activation in LK2-EGFR or LK2-EGFR-S468R cells. Cells were stimulated with EREG or TGFα (100 nmol/L and 10 nmol/L, respectively) and immunoblotted for total and phosphorylated EGFR and AKT (pEGFRY1068 and pAktS473).

Necitumumab binds with high affinity to sEGFR harboring the cetuximab-resistant mutations S468R. A and B, SPR analysis of sEGFR, sEGFR-S468R, and sEGFRd3-S468R binding to immobilized cetuximab (A) and necitumumab (B) Fabs. Normalized equilibrium SPR response plotted as a function of protein concentration were fit to a simple one-site Langmuir binding equation. Data are representative of at least three independent measurements. Mean Kd values with SDs are reported in Supplementary Table S2.

Structure of the sEGFRd3-S468R/Fab11F8 complex. A, A transparent surface representation plus cartoon of the sEGFRd3-S468R/Fab11F8 structure with antibody orange (VH) and yellow (VL) and domain III dark gray. The R468 side chain is in sphere representation colored cyan. Domains I, II, and IV that are not present in the structure are in white (from PDB 1YY9). B, Orthogonal views of the necitumumab paratope, with VH and VL colored as in A, and domain III in gray cartoon. R468 (cyan) sits in a deep hydrophobic cavity between the VH and VL domains. Amino acids lining the cavity are shown in stick representation. Presumed direct and water mediated hydrogen bonds with R468 are indicated with dashed lines.

Comparison of necitumumab and cetuximab paratopes. A, Surface representations of necitumumab and cetuximab (PDB 1YY9) paratopes in the same orientation as in Fig. 3B (right). The electrostatic potential, calculated using the adaptive Poisson–Boltzmann solver (APBS; ref. 49), from −5 kT (red) to +5 kT (blue) is projected on to the surface. R468 (left) and S468 (right) are in cyan stick representation. B, The paratope surfaces are colored to highlight the position of the CDRs. VL CDRs are yellow, CDR H1 pale orange, CDR H2, orange, and CDR H3 brown. Side chains from the CDR H3 of cetuximab that interact with side chains in the VL CDRs are show.

↵c⊗ No binding (Kd value more than 10-fold greater than wild-type). WT binding (Kd value within 10-fold of value for wild-type).

↵dFor counting of true vs false predictions, an EGFR variant is considered an experimental “binder” if the Kd value is less than 10-fold greater than the Kd value for wild-type (). The computational filter is set at ΔΔE < 0. ΔΔE values for true positives [TP, binder () with negative computed energy] and true negatives [TN, nonbinders (⊗) with positive or zero computed energy term] are in black. False predictions (FP and FN) are in gray text. Accuracy is defined as (TP + TN)/(Total number of variants) and precision is defined as TP/(TP + FP).